Aerobic metabolism in living organisms can lead to generation of reactive oxygen species (ROS), which include hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide. Production of ROS can be due to various enzymatic and non-enzymatic processes. In aerobic organisms, ROS are formed from the partial reduction of molecular oxygen to water during oxidative metabolism. Bacterial cells produce endogenous hydrogen peroxide from the dismutation of superoxide or hydroxyl radical as a product of the respiratory chain when oxygen is used as the terminal electron acceptor. Enteric bacteria (e.g. Salmonella typhimurium and E. coli) encounter toxic levels of hydrogen peroxide produced by macrophages during engulfment.
Under normal conditions, ROS may play an important role in different biological processes. However, when ROS are excessively produced under certain unusual conditions, they can cause oxidative damage to DNA, proteins and lipids.
ROS have been implicated in the pathogenesis of many different disease situations as well as harmful conditions. These include aging, AIDS, atherosclerosis, cancer, cataracts, congestive heart failure, diabetes, inflammatory disorders, rheumatoid arthritis, and neuro-degenerative diseases such as Alzheimer's, Parkinson's, multiple sclerosis, and Down's syndrome, in addition to exposure to pollutants and ionizing irradiation.
Living organisms have developed different ways of coping with the ROS. The capacity of enzymatic or non-enzymatic antioxidants to quench the ROS can help cells to defend against the oxidative stress. Therefore, antioxidants have been linked to and used for disease prevention.
Antioxidants may be proteins, such as ferritin, lactoferritin and transferritin, or enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. Nonenzymatic antioxidants may be macromolecules, such as albumin, copper-binding ceruloplasmin and hemoglobin, or small molecules which may be water-soluble antioxidants, such as vitamin C, uric acid and bilirubin or lipid-soluble antioxidants, such as vitamin E, carotenoids, retinoids and ubiquinol-10.
However, these natural defenses can be overwhelmed in many pathological states. More potent antioxidants should be supplemented to deal with the oxidative stresses. Screening and assay methods are needed to identify potent antioxidants; but the current methods are both time-consuming and expensive. In addition, they cannot measure the intracellular antioxidant activities, which are more relevant to biological applications. Therefore, a simple method that can measure the intracellular antioxidant activities and hence can be used to search for better oxidant scavenging molecules is of great importance in the pharmaceutical and nutraceutical fields.
The public has shown an increasing interest in the natural antioxidants contained in dietary supplements, as antioxidants can give health benefits by preventing oxidative damage caused by ROS. Standardized assays to assess antioxidant activities and distinguish different antioxidants are useful. Such assay methods are useful to properly assess and label antioxidant products. Such assays are also useful for measuring activities of antioxidants for use as food supplements, natural products and drugs.
Farr (U.S. Pat. Nos. 5,585,252, 5,811,231 and 5,589,337) have described use of stress promoters fused to reporter genes to determine toxicity.
Catalase is a protein antioxidant. Catalases catalyze the dismutation of hydrogen peroxide to water and oxygen. The primary role for catalases is to protect the cells against the damage caused by reactive oxygen species to cellular components, including nucleic acids, proteins and cell membranes. Catalases have been implicated to be important for the survival of some pathogenic bacteria during infection and even for the life span of a multi-cellular organism.
Agrobacterium tumefaciens is a soil-borne plant pathogen that causes crown gall tumors on many plant species. A chromosomal gene katA encoding a catalase has been identified that is involved in detoxification of H2O2 released during Agrobacterium-plant interaction (Xu and Pan, 2000).